Keratan sulfate proteoglycans (KSPGs) are extracellular matrix molecules that appear to establish boundaries for axonal growth in the developing brain and spinal cord. In vitro studies confirm that KSPGs define inhibitory boundaries to extending neurites. The aim of the current study was to investigate whether KSPGs are expressed after spinal cord injury (SCI) and thereby might act as potential inhibitors of axonal growth. Adult Fischer 344 rats were subjected to spinal cord lesions, and the temporal and spatial expression of KSPGs was examined using the 5D4 monoclonal anti-KSPG antibody. In the intact spinal cord, a subpopulation of microglia expressed 5D4-KSPG throughout the white and gray matter. Within 24 hr of injury, 5D4-KSPG immunoreactivity substantially increased and appeared on cellular profiles in close proximity to the spinal cord lesion site, peaking 3 d after injury. Double immunolabeling revealed that 5D4-KSPG expression arose from multiple cell types at the lesion site, including reactive microglia, macrophages, and oligodendrocyte progenitors. Astrocytes were not identified as a source of 5D4-KSPG. The robust and extensive production of 5D4-KSPG at sites of SCI precedes the expression of other putatively inhibitory proteoglycan molecules such as chondroitin sulfate proteoglycans. This is the first demonstration that KSPGs are expressed after SCI in a temporal and spatial relationship that could exert an early and important role in modulating axonal growth after SCI.